Cataracts are principally a disease of aggregation and subsequent precipitation of specific lens structural proteins (crystallins), Cataracts may be induced by genetic mutations, or may be associated with aging. In the course of cataract development, the aggregated and cross-linked crystallin multimers become so large that they finally become water insoluble and cause lens opacity. This proposal has the overall goal to determine the molecular mechanisms of congenital nuclear cataract development and of age-related cortical cataract development. We have generated two mouse models, a conditional ?A3/A1 knockout (?A3cKO) mouse model and an ?A-N101D transgenic mouse model. Characterization of the ?A3cKO mice by the PI has established their validity as a model of congenital nuclear cataract development, and will serve as a model system to better understand the molecular mechanism of ?A3-mutation-induced pediatric cataracts. Characterization of the ?AN101D mice by the PI has established its validity as a model of deamidation-induced age- related cortical cataract development. Based on our results and results of others, our central hypothesis is that: (A) Congenital cataract in the ?A3-cKO mouse model is caused by a dysfunctional degradation process (autophagy) due to the absence of ?A3 as a lysosomal resident protein, accelerated calpain-induced proteolysis of lens proteins, and disruption of protein-protein interactions; and (B) The age-related cortical cataract in the ?AN101D mouse model is caused by intracellular ionic imbalance due to increased membrane binding of ?AN101D, leading to calpain activation, changes in cellular junctions and actin filaments due to altered RhoA, Cdc42, and Rac activities, and the loss of protein-protein interactions. We hypothesize that in cataractogenic mechanisms of both models, the aggregation of truncated crystallin fragments produced by calpain plays a major role. We plan to test our central hypothesis in these mouse models in three aims: (A) Aim1: What is the role of ?A3 crystallin in autophagy blockade (in ?A3KO mice), and what is the role of calpain activation and protein-protein interactions in the cataractogenic mechanisms of both ?A3cKO and ?AN101D mice? (B) Aim 2: What is the molecular mechanism of cataract development in transgenic ?AN101D mice? (C) Aim 3: What are roles of crystallin fragments generated by proteolysis of lens proteins by calpain in cataract development of ?A3cKO and ?AN101D mouse models? We expect that the findings will provide insights to guide us toward improved therapeutic intervention to delay or cure congenital pediatric and age-related cataracts.